| Objective:The fundamental mechanism of acute lung injury (ALI) is the inflammation that is part of the dody's immune response to a variety of local or systemic stimuli. Lipoxins (LXs) are important endogenous lipids that mediate resolution of inflammation. The airway epithelia are the prime target of inhaled toxicants that also create the first physical barrier against these noxious substances. Lipopolysaccharide (LPS) is an important trigger of ALI through inducing a massive accumulation of neutrophils, production of reactive oxygen species (ROS) and releasing of cytokines in lungs. Basic on our previous work, present study further studied on the protective role of LXA4 in LPS-induced mice ALI and underlying mechasnisms, especially the role of airway epithelium. We explored the effect of LXA4 on LPS-induced damage of E-cadherin and permeability in airway epithilium. At the same time, the activation and the translocation of NF-E2-related factor 2 (Nrf2) were also investigated.Methods:(1) Establishment of LPS-inhalation induced ALI model:All mice were anesthetized with pentobarbital (70 mg/Kg i.p.) and exposed either to aerosolized 0.9% saline (control or LXA4 group) or 200 mg/ml LPS in saline (LPS group or LPS+LXA4 group) for 20 min under a laminar flow hood, at the flow rate of 1 ml/min. One hour after the exposure, the mice in the LPS+LXA4 group and LXA4 group received 100 mg/kg LXA4 by tail vein injection. All in vivo experiments were conducted 8 hours after LPS inhalation.(2) 16HBE cell treatment:human airway epithelium cell line 16HBE was treated with 100 ng/ml LPS and/or 200 nM LXA4 for 24 h to observe the expression of E-cadherin and the production of ROS.(3) Low-temperature imaging (cryo-imaging) assays:8 h after ALI model establishment, the mice were anesthetized with pentobarbital (70 mg/kg i.p.) and slowly immersed in liquid nitrogen until analysis. The frozen lung tissue was sectioned sequentially in the longitudinal direction then the autofluorescence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was excited to probe the lung mitochondrial redox status in mice.(4) Fluorescence resonance energy transfer (FRET):After two plasmids expressing ECFP-Kelch-like ECH-associated protein 1 (Keap1) or EYFP-Nrf2 protein had been co-transfected into HBE cells for 24 h, FRET was performed using a Zeiss LSM710 confocal microscope to evaluate the dissociation process of Nrf2 and Keapl, a necessary step of Nrf2 activation.(5) Nrf2 inhibitor SB203580 and pDN-Nrf2 plasmid treatment:After 5 umol/kg SB203580 was injected via tail vein for 30 min (LPS+LXA4+SB203580 group), the mice were exposed to 200 ug/ml LPS in saline by aerosolization for 20 min, the LXA4 (100ug/kg) was injected i.v.1 h later (LPS+LXA4 group or LPS+LXA4+SB203580 group). The 16HBE cells were transfected with or without pDN-Nrf2 plasmid, and 24 h later treated with 100 ng/ml LPS and/or 200 nM LXA4 with or without 20 uM SB203580 for an additional 24 h. The experiment was designed to demonstrate that pSer40 Nrf2 was the main form of Nrf2 contributing to the elevation of nuclear Nrf2 after LXA4 treatment and to explore whether Nrf2 was involved in the protective effect of LXA4 on E-cadherin expression.Results.1?LXA4 effectively inhibited LPS-induced ALI in mice;2?LXA4 effectively protected airway epithelium from LPS induced damage;3?LXA4 blocked LPS-induced ROS elevated production in lung of ALI mice and 16HBE cells;4?LXA4 regulated the lungs mitochondrial from exidative status to reductant status in LPS-treated mice;5?LXA4 promoted Nrf2 Ser40 phosphorylation and its activation in 16HBE cells;6?The regulation of LXA4 on the expression of E-cadherin in 16HBEs was significantly blocked by transfection of pDN-Nrf2, a dominant negative mutant Nrf2 plasmid;7?The relief of LPS-induced ALI in LXA4 treated mice was reversed by selective Nrf2 inhibitors.Conclusion:These results provide a new mechanism by which LXA4 inhibits LPS-induced ALI through Nrf2-mediated E-cadherin expression. |
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